The role of deubiquitinating enzyme USP22 in human somatic cell reprogramming

Human somatic cells can be reprogrammed to induced pluripotent stem cells (iPSC) by overexpressing OCT4, SOX2, KLF4 and MYC (OSKM). There are cell intrinsic barriers to reprogramming. We conducted a CRISPR-Cas9-mediated knockout screen during reprogramming to reveal chromatin pathways acting as barriers to reprogramming. Other than DNMT3A and EP300 which were already known to be barriers to reprogramming, this screen revealed a barrier role for USP22 during reprogramming. In this thesis, I validated the barrier role of USP22 in reprogramming by loss-of-function assay. In addition to this, overexpression of various USP22 mutants revealed that USP22 deubiquitinase activity or its integration into the SAGA complex does not affect reprogramming. Interestingly, CRISPR-Cas9-mediated knockout of SAGA deubiquitinase members, ATXN7L3 and ENY2 had no positive impact on reprogramming efficiency. To investigate the effect of USP22 on human pluripotency, I obtained single USP22 knockout clones. These clones expressed pluripotency markers, contributed to tissues from three germ layers when subjected to teratoma formation assay and showed normal karyotyping as in control pluripotent stem cells. To understand the defects in specific lineage specifications, these clones were subjected to in vitro embryoid body formation assay. There seem problems in pluripotency exit as revealed by unsuccessful downregulation of pluripotency markers such as OCT4 and SOX2. Furthermore, mesoderm and endoderm differentiation defects were observed in one of USP22 knockout clones compared to wild-type clones as judged by the lower expression levels of marker genes. To gain more mechanistic insight on the USP22 loss-mediated enhanced reprogramming, we performed an RNA-Seq experiment. USP22 knockout and wild-type fibroblasts were reprogrammed by OSKM expression and on day 6 of reprogramming RNA-Seq was performed. Expectedly, we observed that development-related genesets were negatively enriched whereas pluripotency-related genesets were positively enriched by USP22 loss. SOX2 target geneset was among positively enriched genesets and we hypothesized that USP22 loss activates endogenous pluripotency network earlier during reprogramming to increase its efficiency. To test this hypothesis, we collected RNA at different days of reprogramming and revealed that endogenous SOX2 levels increased up to 3-fold upon USP22 loss during reprogramming. These results show that USP22 acts as a barrier to reprogramming by suppressing endogenous pluripotency network independent from its catalytic activity and SAGA incorporation.